PROJECT SUMMARY/ABSTRACT We propose testing the hypothesis that shifts in the human cytotrophoblast (CTB) epigenome early in pregnancy are integral to their normal differentiation, formation of the placenta and pregnancy success. CTB fate decisions establish placental structure and thereby function. In one pathway, CTBs fuse to form the syncytiotrophoblasts (STBs) that cover the surface of chorionic villi. These cells produce hormones and exchange myriad substances between the mother and the embryo/fetus. In the other differentiation pathway, CTBs emigrate from the chorionic villi and invade the uterus, anchoring the placenta to the mother. In the process, they tap into the resident arteries and veins, establishing blood flow to the intervillous space. Recently we employed sequencing approaches to profile the CTB epigenome in late 2nd trimester and at term. The data revealed a unique pattern of global hypomethylation punctuated by megabase domains of even deeper valleys of hypomethylation. In comparison, other human embryonic, fetal, and adult genomes were highly methylated. Unexpectedly, H3K9me3 occupancy precisely overlapped domains with more pronounced DNA hypomethylation and repressed transcription. Also, we discovered significant gestational age-related alterations in the CTB epigenome. During the late 2nd trimester-to-term interval, substantial genome-wide increases in DNA methylation were accompanied by depletion of H3K9me3 and H3K4me. Surprisingly, we also found changes in histone abundance at the end of the 1st trimester, i.e., a substantial decrease in H3K27me3 and H3K4me1 signals. Work from other investigators suggests parallel increases in DNA methylation of placental promoters over the same period. Thus, the CTB epigenome appears to be evolving throughout pregnancy with significant changes early as well as later in gestation. To understand the functional consequences of these shifts, we propose two Specific Aims. First, we will investigate the relationship between alterations in the CTB epigenome and transcriptome at the end of the 1st trimester of pregnancy. Changes in DNA methylation and key histone modification profiles will be intersected with expression data to identify pathways and their drivers that are modulated. Second, we will test the functional significance of the drivers in our in vitro models of CTB differentiation, which enable interrogating formation of STBs or invasive extravillous CTBs. The most innovative aspect of this project is the intent to build on our recent discovery of the unique nature of the human CTB epigenome, which exhibits substantial shifts over gestation. The significance lies in the importance of CTB differentiation to placental development and function. Our goal is to achieve a new level of understanding about the mechanisms that are involved, information that has translational value in terms of gaining new insights into failures in these processes. We reason that they may be associated with some cases of infertility and 1st trimester losses or lie at the root of pregnancy complications associated with faulty placentation, e.g., preeclampsia intrauterine growth restriction.